1. Field of the Invention
The present invention relates generally to the field of cellular factors (proteins) which bind nucleic acids, particularly those factors which bind the nucleic acid region previously found to bind a factor or group of factors known as the nuclear factor of activated T cells (the NFAT region). The gene which encodes the cellular factor is also related to the field of the present invention. Recombinant vectors and host cells including the gene for the cellular nucleic acid binding factor is also related to the present disclosure. The invention also relates to the field of methods for regulating the expression of cellular and viral genes including lymphokine genes such as IL-2 and HIV gene expression, and to methods for treating HIV and AIDS infection.
2. Description of the Related Art
The human immunodeficiency virus (HIV) is the causative agent of AIDS. In common with other retroviruses, HIV contains two long terminal repeats (LTRs) and three conserved genes, namely gag, pol, and env. Once HIV-1 integrates into the host cell genome, its gene expression is regulated by cellular transcription factors in a manner similar to that of endogenous cellular genes. The activities of several of the cellular transcription factors which bind to the HIV-1 LTR are altered by parameters such as activation or differentiation of lymphocytes or macrophages, the action of lymphokines, and alterations of signal transduction pathways. Thus, HIV is subject to many of the same regulatory signals that are important in controlling cellular gene expression.
A number of elements in the HIV long terminal repeat (LTR), including SP1, TATA, and TAR are involved in regulating gene expression in a variety of cell lines (Garcia et al., 1989). At least two other regulatory regions have been determined to be important for activation of HIV-LTR gene expression in activated T-lymphocytes. One is the enhancer region extending from nucleotides -103 to -78 which contains two NF-kappa B motifs (Nabel & Baltimore, 1987; Tong-Starksen et al., 1987; Gaynor et al., 1988). The other is a region extending from nucleotides -283 to -195 which contains several motifs with homology to a critical regulatory domain in the interleukin-2 (IL-2) promoter (Fujita et al., 1986; Shaw et al., 1988; Crabtree, 1989).
The referenced regulatory domain of the IL-2 promoter is characterized as a motif which is a positive acting element. This positive acting element binds a cellular factor known as the nuclear factor of activated T-cells (NFAT) whose binding is strongly induced in activated T-cells (Fujita et al., 1986; Shaw et al., 1988; Crabtree, 1989). NFAT is composed of two subunits, a constitutive nuclear factor and an inducible T-cell factor. In the HIV-LTR, this region containing NFAT-like motifs is important for increasing HIV gene expression in activated T-cells (Siekevitz et al., 1987), but also appears to have negative regulatory effects on HIV gene expression in the presence of tat (Siekevitz et al., 1987; Lu et al., 1990).
Certain poorly characterized cellular proteins have been reported to bind to NFAT-like regulatory motifs. In activated T-cells, UV crosslinking studies are said to demonstrate that three cellular proteins of 90 kD, 45 kD, and 25 kD bind to the NFAT motif in the IL-2 promoter (Randak et al., 1990). Cyclosporin inhibits the binding of each of these proteins, suggesting that multiple proteins with a similar pattern of regulation may bind to this region. Recently, a cellular factor restricted to B lymphocytes and macrophages, known as PU.1 (Pettersson & Schaffner, 1987; Klemsz et al., 1990), which has strong homology to the ets proto-oncogene, was found to bind to a related motif found in SV 40 (Karim et al., 1990). Thus, different DNA binding proteins may be capable of binding to these purine-rich motifs.
A macrophage and B-cell-specific transcription factor, PU.1, with homology to the ets oncogene, also binds to a purine-rich sequence GAGGAA identical to that found in the NFAT motif (Klemsz et al., 1990). This protein has been demonstrated to have positive effects on SV40 gene expression (Karim et al., 1990).
Despite the studies discussed above, there remains relatively little documented information on cellular factors which are capable of binding to the NFAT-like motifs in the HIV-LTR. In particular, the mechanisms by which this region can exert both positive and negative regulation on HIV gene expression remain to be elucidated.
Disturbances of T cell regulation have been implicated in immune system pathology such as immunodeficiency and autoimmune disorders, and may also play an important role in the development of AIDS following latent HIV infection. The identification of NFAT regulatory factors would represent an important advance in the knowledge of IL-2 and HIV gene regulation, and would thus further the understanding of the processes underlying immune homeostasis, disease, and AIDS. Such a discovery may also open up new avenues of clinical investigation, including the development of AIDS- and immune-therapeutic agents.
The nucleotide sequence of the HIV-LTR reveals several purine-rich motifs between nucleotides -283 and -195 having a homology to an element in the IL-2 promoter (Fujita et al., 1986). This element is a binding site for the cellular factor, NFAT, whose binding is induced in activated T-cells (Fujita et al., 1986; Shaw et al., 1988; Crabtree, 1989). Indeed, in addition to regulation by general transcription factors, HIV gene expression is likely regulated by NFAT (Crabtree, 1989). In the HIV LTR, the -283 to -195 region containing the NFAT-like motifs is important for increasing HIV gene expression in activated T-cells but it has little effect on basal gene expression (Siekevitz et al., 1987). However, in the presence of tat it appears to have negative regulatory effects on HIV gene expression (Rosen et al., 1985).
DNase I footprinting demonstrates the binding of cellular factors from -254 to -216 in the HIV-LTR with extracts prepared from activated but not resting T-cells (Shaw et al., 1988; Crabtree, 1989). An increase in NFAT binding precedes the activation of both IL-2 and HIV gene expression in stimulated T-cells (Shaw et al., 1988; Crabtree, 1989). The induction of NFAT binding requires new protein synthesis (Shaw et al., 1988; Crabtree, 1989), and its binding is inhibited by cyclosporin, an inhibitor of the enzyme cyclophilin (Emmel et al., 1989). However, the production of NFAT is not completely restricted to activated T-cells since low levels of NFAT binding are also detected in resting T-cells (Novak et al., 1990).
Multiple cellular proteins have also been demonstrated to bind to the NFAT-motifs in the IL-2 promoter (Randak et al., 1990). As already noted, the binding of NFAT is strongly induced by T-cell activation.
It appeared to the present inventors that specific proteins which bind to the NFAT-like motif were important in regulating HIV gene expression. The inventors therefore proceeded to examine the various characteristics which such an NFAT--binding factor would have in their attempt to isolate and characterize a specific and unique binding factor. Such a protein/factor which was capable of binding an NFAT-motif on the HIV or which would bind the protein which binds the NFAT motif region of the HIV-LTR, would provide a specific and unique method for inhibiting HIV-1 gene expression, and thereby the method for inhibiting the pathologies in humans which HIV-1 infection causes, such as AIDS and ARC.